The specific aim of this proposal is to understand the mechanisms by which splicing of introns from precursor messenger RNA (pre-mRNA) is regulated. Splicing is a highly efficient process which in many instances involves the regulated use of alternative splice sites. Splicing is done by a large multicomponent ribonucleoprotein complex, the spliceosome, which is formed by an ordered assembly of its subunits, the snRNPs, onto the nascent pre- mRNA. This assembly defines the intron boundaries at which splicing will occur. l have previously identified, isolated and characterized a family of pre-mRNA splicing factors which are required for the earliest step of spliceosome assembly. I named these the SR proteins for the serine and arginine rich C-terminal domain they all contain. There are at least six SR protein family members, each of which has been highly conserved in evolution in metazoans. I have shown that the presence of at least one of the SR protein family members is required for pre-mRNA splicing, and that different family members have distinct abilities to promote splicing at alternative splice sites. These alternative splicing factors are required for their first step of spliceosome assembly, binding of the U1 snRNP to the pre-mRNA, and different SR proteins have distinct abilities to recruit U1 snRNP to alternative 5' splice sites. Our understanding of the mechanisms that regulate spliceosome assembly will be furthered by an understanding of how the SR proteins function in this regulation. In order to accomplish this, we will study the interactions of SR proteins with the pre-mRNA to identify signals on the nascent transcripts through which SR proteins act to direct alternative splice site usage. We will analyze the different domains of the SR proteins through domain swap experiments to understand the functions of the different distinct domains which SR proteins share. We will identify protein factors which interact specifically with the different SR proteins in order to understand the mechanism by which SR proteins promote spliceosome assembly and whether they function at other steps of spliceosome assembly and splicing.